1. Field of the Invention
The present invention relates to a complex of enzyme, protein and carrier prepared by conjugating a protein with a specific binding potency to other substance(s) onto an enzyme covalently conjugated to a carrier and the complex is utilized for immunoassay such as immunohistochemistry and enzyme immunoassay.
2. Description of the Related Art
Owing to the recent progress in immunochemistry, immunoassay capable of detecting a trace amount of a substance at a high sensitivity by using an antigen-antibody reaction has been used widely. Currently, two fields of general types of immunoassay are immunohistostaining and enzyme immunoassay.
Immunohistostaining is means for detecting a specific antigen on a tissue with an antibody specifically recognizing the antigen. Generally, an antibody recognizing a specific antigen is subjected to a reaction on a thin section sliced from a block prepared by fixing a tissue and then embedding the tissue in paraffin; by examining the presence or absence of the reacted antibody, the presence of the antigen can be determined. The antibody allowed to first react with the antigen is generally called primary antibody. When a substance emitting a signal detectable visually or with an apparatus is conjugated to the primary antibody, the intensity of the signal indicates the amount of the primary antibody, which corresponds in turn to the amount of the antigen on the section. As the substance emitting the signal for attaining the purpose, fluorescent substance and enzyme may be mentioned. At an early development stage of immunohistostaining, fluorescent substance was used as a substance emitting such signal. In that case, fluorescent microscope was necessary for detecting fluorescence.
Subsequently, enzyme-labeled antibody method was developed by Nakane et al., which enabled the analysis of stained image with optical microscope. Currently, enzyme is generally used as a substance emitting signal. For effecting immunohistostaining, a color reaction corresponding to the activity of an enzyme if conjugated to a primary antibody can be effected by adding a chromogenic substance to the enzyme, and the color reaction corresponds to the amount of the antibody, namely the amount of the antigen present on the tissue. However, generally, a sufficient sensitivity can never be recovered by the method.
The method most commonly used currently is called streptavidin-biotin method (SAB method), namely means for amplifying the signal of a primary antibody bound to an antigen, thereby detecting the signal. The method comprises first subjecting a primary antibody recognizing a specific substance on a tissue section to reaction therewith. Then, a secondary antibody binding to the primary antibody is subjected to reaction therewith. Generally, the secondary antibody is a polyclonal antibody which recognizes and bonds to the primary antibody thereto. Accordingly, plural molecules of the secondary antibody are bound to the primary antibody. Plural molecules of biotin are preliminarily conjugated to each molecule of the secondary antibody. An enzyme-conjugated streptavidin (enzyme reagent) is allowed to react with the primary antibody-biotin-conjugated secondary antibody complex. It is known that streptavidin can strongly be bound to biotin. Therefore, a complex of primary antibody—biotin -conjugated secondary antibody—enzyme-conjugated streptavidin is formed. Because plural molecules of the biotin-conjugated secondary antibody conjugate are bound to each molecule of the primary antibody and plural molecules of the enzyme-conjugated streptavidin conjugate are bound to each molecule of the biotin-conjugated secondary antibody conjugate according to the method, consequently, the amount of the enzyme bound indirectly to the primary antibody can be increased markedly. As a result, the antigen on the tissue section can be detected at a high sensitivity.
As described previously, the SAB method is an excellent method capable of producing a more intense signal because a great many molecules of an enzyme are ultimately bound to the primary antibody bound to the antigen. However, from another standpoint of the procedures, three steps of procedures, namely reaction of primary antibody, reaction of secondary antibody, and reaction of enzyme reagent, are required to be carried out. As described previously , the SAB method includes many steps and cannot be evaluated to be satisfactory in the aspects of clinical practice and the like, demanding rapidity and simplicity along with accuracy. Thus, it is expected that the SAB method is improved.
As another means of general immunoassay, enzyme immunoassay (EIA) is known. Typical principle and procedure of EIA are as follows. First, an antibody recognizing a substance desired to be assayed is immobilized on a carrier such as polystyrene bead or microplate. After subsequently blocking the carrier with protein such as albumin, a solution (sample) containing a substance (antigen) as an intended assay object is then added. Thereafter, an antigen-recognizing antibody conjugated with an enzyme (enzyme-labeled antibody) is added. In other words, two antibodies interpose the antigen therebetween. Then, an excess of the enzyme-labeled antibody is washed off; a chromogenic substrate of the enzyme is added for color development. Because the amount of the antigen depends on the activity of the enzyme, the concentration of the antigen in the sample can be determined by comparison with the color development of a sample containing antigen at preliminarily known concentration. A number of factors are responsible for the sensitivity of enzyme immunoassay, and the quality of the enzyme-labeled antibody is one of the significant factors. More specifically, it is thought that a more intense signal can be obtained when the enzyme-labeled antibody has many molecules of the enzyme, whereby the antigen can be assayed at a high sensitivity.
As described previously, the quality of the enzyme-labeled antibody is very important for immunoassay and influences markedly the sensitivity of assay system or the number of the steps included in the procedure. As illustrated below, many attempts have been made in order to obtain an enzyme-labeled antibody that achieves a high sensitivity. Many of them comprise conjugating many molecules of an enzyme and an antibody to a carrier.
In Japanese Patent Application Publication JP-A 63-503138 (1988), an antibody was conjugated to a carrier conjugated with a derivative of a detectable label such as drug, toxin, chelator and boron adduct. As the carrier, is used aminodextran; first, a drug such as methotoxate is conjugated to aminodextran. Thereafter, aldehyde group generated from the oxidation of the sugar chain of the antibody with sodium periodate was allowed to react with the amino group of aminodextran, followed by reduction with sodium cyanoborohydride to effect covalent bond, to prepare a complex of the drug, the antibody, and aminodextran.
In Japanese Patent Application Publication JP-A 3-158758 (1991), dextran was oxidized with sodium periodate; the resulting aldehyde group was allowed to react with the amino group of alkaline phosphatase and antibody, followed by reduction with sodium borohydride, to prepare a complex of the enzyme, the antibody and dextran.
In Japanese Patent Application Publication No. 6-509167 (1994), divinyl sulfone was reacted with a polymer such as dextran to introduce the vinyl group therein, followed by reaction with an enzyme and an antibody, thus a complex of the enzyme, the antibody and dextran being prepared.
All the complexes prepared by these methods were complexes formed by conjugating directly two substances to polymer. All the complexes prepared by these methods are said to show better performance, compared with direct conjugating of the two substances with no use of any carrier. However, these methods have the following drawback. More specifically, because the amount of substances capable of conjugating to a carrier in the case that two substances are to be conjugated to the carrier is finite, the larger the amount of one substance is conjugated thereto, the smaller the amount of the other substance is conjugated thereto. If the drawback can be overcome, a complex with better performance is possibly obtained.